CN110327347B - Application of G-749 in preparing antifungal medicine - Google Patents

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of G-749 in preparation of an antifungal medicine. In vitro cell experiments show that G-749 has antifungal and even fungicidal effects to different degrees on drug-resistant Candida albicans 103, Candida albicans SC5314, Cryptococcus neoformans, Candida krusei, Candida parapsilosis, Candida glabrata and Candida tropicalis, so G-749 can be used as an antifungal drug. At present, the antifungal spectrum is wide clinically, drugs with antifungal activity are few, and under the conditions that the clinical fungal drug resistance is more and more common and the drug resistance degree is more and more serious, the protein kinase inhibitor G-749 has the characteristics of wide antifungal spectrum and antifungal activity, and provides a new treatment way for effectively treating the infection of fungi and even drug-resistant fungi clinically.

Description

Application of G-749 in preparing antifungal medicine

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of a protein kinase inhibitor 8-bromo-2- [ (1-methyl-4-piperidyl) amino ] -4- [ (4-phenoxyphenyl) amino ] pyrido [4,3-d ] pyrimidine-5 (6H) -ketone (commonly known as G-749) in preparation of antifungal medicines.

Background

G-749 is a novel FMS-like tyrosine kinase 3 (Flt 3) inhibitor, and G-749 exhibits antiproliferative activity by inducing apoptosis in leukemia cells. Of the drug candidates currently in clinical research, about 20% to 30% of the drugs are protein kinase inhibitors. Although protein kinase inhibitors have been widely studied in the fields of tumors, diabetes, rheumatism, and the like, and many studies have been reported in recent years in terms of resistance to bacterial infection, there have been few studies in the field of resistance to fungal infection. At present, no report of the antifungal effect of G-749 is found at home and abroad.

Disclosure of Invention

The invention aims to provide application of G-749 in preparing antifungal medicines.

The invention is realized by the following technical scheme:

g-749 is used in preparing antifungal medicine.

Preferably, the fungus is a susceptible fungus strain or a resistant fungus strain.

Further, the sensitive fungus strain is sensitive to fluconazole, and the drug-resistant fungus strain is drug-resistant fungus strain resistant to fluconazole.

Preferably, the fungus is candida krusei (ATCC2340), candida glabrata (ATCC2281), cryptococcus neoformans (32609), drug-resistant candida albicans (103), sensitive candida albicans (SC5314), candida parapsilosis, or candida tropicalis.

Preferably, the MIC value of G-749 is 16. mu.g/mL.

An antifungal medicine is prepared from G-749 and medicinal adjuvants.

Compared with the prior art, the invention has the following beneficial technical effects:

in vitro cell experiments show that G-749 has antifungal and even antifungal effects to different degrees on drug-resistant Candida albicans (103), sensitive Candida albicans (SC5314), cryptococcus neoformans, Candida krusei, Candida parapsilosis, Candida glabrata and Candida tropicalis, so that G-749 can be used as an antifungal drug. The invention opens up a new application of G-749, and provides a new candidate drug for preparing antifungal drugs and treating fungal infection. At present, the clinical antifungal spectrum is wide, the number of drugs with antifungal activity is very small, and under the conditions that the clinical fungal drug resistance is more and more common and the drug resistance degree is more and more serious, the characteristics of the broad antifungal spectrum and the antifungal activity of G-749 provide a new treatment way for the clinical high-efficiency treatment of the infection of fungi and even drug-resistant fungi.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The invention provides an application of G-749 in preparing antifungal drugs, wherein the application comprises the following steps: the G-749 is a protein kinase inhibitor (Flt3 inhibitor) 8-bromo-2- [ (1-methyl-4-piperidinyl) amino ] -4- [ (4-phenoxyphenyl) amino ] pyrido [4,3-d ] pyrimidin-5 (6H) -one with CAS number 1457983-28-6.

In the present invention, the fungi include Candida krusei (ATCC2340), Candida glabrata (ATCC2281), Cryptococcus neoformans (32609), drug-resistant Candida albicans (103), sensitive Candida albicans (SC5314), Candida parapsilosis, and Candida tropicalis.

In the invention, the drug-resistant strain is resistant to fluconazole, and the sensitive strain is sensitive to fluconazole.

Example 1 Effect of G-749 on different fungi

Materials and methods

1. Reagent testing:

g-749(CAS number 1457983-28-6): purchased from Shanghai Danquan medicine science and technology Limited and has a purity of more than or equal to 99 percent.

And (3) fluconazole: sigma Inc., batch No. 036M 4709V.

Dimethylsulfoxide (DMSO): tianjin, Nature chemical reagents, Inc.

Each reagent was prepared in DMSO and stored at-20 ℃. Before the experiment, the medicine is taken out and placed in a 35 ℃ incubator to be melted and fully mixed.

2. The strain is as follows:

the strains used were: comprising 1) sensitive strains: sensitive candida albicans (SC5314), candida parapsilosis (22019), cryptococcus neoformans (32609); 2) drug-resistant strains: drug-resistant Candida albicans (103), drug-resistant Candida tropicalis, naturally drug-resistant Candida krusei (ATCC2340), and naturally drug-resistant Candida glabrata (ATCC 2281). The strains are donated to the subject group of Jiangyuan teachers at the second medical university, and are identified by morphology and biochemistry.

All strains for experiments are subjected to scratching activation on a Saburg glucose agar (SDA) culture medium, and after candida albicans, candida tropicalis, cryptococcus neoformans and other cocci are cultured for 2-3 days at 35 ℃, monoclones are respectively picked and scratched again for activation, and the monoclones obtained in the second time are taken and placed on an SDA inclined plane, and are stored for later use at 4 ℃ after being cultured by the method.

3. Culture solution:

1) RPMI1640 liquid culture medium:

RPMI1640(Gibco)10g，NaHCO₃2.0g and 34.5g (0.165mol) of morpholine propanesulfonic acid (Sigma), adding 900mL of triple distilled water for dissolution, adjusting the pH to 7.0 by 1mol/L of NaOH, fixing the volume of the triple distilled water to 1000mL, filtering and sterilizing by a 0.22 mu m microporous filter membrane (Millipore), subpackaging, and storing at 4 ℃ for later use.

2) Sandcastle glucose agar solid medium (SDA):

dissolving 10g of peptone and 40g of glucose in 900mL of triple distilled water, adding 18g of agar, adjusting the pH to 7.0, metering the volume to 1000mL of triple distilled water, sterilizing under high pressure (121 ℃, 20min), and storing at 4 ℃ for later use.

3) YEPD culture solution:

dissolving yeast extract 10g, peptone 20g and glucose 20g in 900mL of triple distilled water, diluting to 1000mL of constant volume, sterilizing under high pressure (121 ℃, 20min), and storing at 4 ℃ for later use.

4. The instrument equipment comprises:

GHP-9080 type water-proof constant temperature incubator (Shanghai-Hengscientific instruments Co., Ltd.);

THZ-98C type constant temperature shaking box (Shanghai-Hengchun scientific instruments Co., Ltd.);

SW-CT-IF model superclean bench (Suzhou Antai air technologies, Inc.).

G-749 and fluconazole stock solutions:

the purchased G-749 is weighed and dissolved in a proper amount of dimethyl sulfoxide to prepare 6.4mg/mL of medicine solution, and the medicine solution is put into a refrigerator for standby at the temperature of-20 ℃.

The fluconazole is dissolved by DMSO to prepare a concentration of 6.4mg/mL, and the mixture is stored at the temperature of minus 20 ℃ for later use.

6. Preparing bacterial liquid:

before the experiment, a small amount of each globular fungus is picked from an SDA culture medium stored at 4 ℃ by using an inoculation ring, and is respectively inoculated into 1mL of YEPD culture solution, and is subjected to shaking culture at the speed of 200rpm at the temperature of 35 ℃ for 16h, so that the fungus is in the later period of exponential growth. Then adding each bacterial liquid to 1mL YEPD culture solution, activating again by the above method, culturing for 16h, counting by using a blood cell counting plate, and adjusting each bacterial liquid concentration to 1 × 10 with RPMI1640 culture solution³～5×10³CFU/mL。

7. Preparing a drug sensitive plate:

taking each strain separately one sterile 96-well plate, and adding 100 μ L of RPMI1640 liquid culture medium into each row of No. 1 wells as blank control; adding 100 mu L of the freshly prepared bacterial liquid into each of the No. 3-12 holes; adding 200 mu L of bacteria solution into No. 2 pore; no. 12 wells contained no drug, and 100. mu.L of inoculum was added as a positive growth control. G-749 or fluconazole with the mother liquor concentration of 6.4mg/mL is added into the No. 2 hole of each row, and each volume is 2 mu L.

The final G-749 and fluconazole concentrations in each well of No. 2-11 wells were 64. mu.g/mL, 32. mu.g/mL, 16. mu.g/mL, 8. mu.g/mL, 4. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL, 0.125. mu.g/mL by 10-fold dilution. The DMSO content in each well was below 1%. The drug sensitive plates were cultured in a thermostat at 35 ℃.

MIC value determination:

the culture was carried out at 35 ℃ for 24 hours in an incubator, and the results of the experiment were observed to determine the MIC values. After the observation is finished, the culture medium is placed back into the constant temperature incubator for continuous culture. When the MIC value of the drug exceeds the measured concentration range, counting is carried out according to the following method: when the MIC value is higher than the highest concentration of 64 mu g/mL, the MIC value is calculated to be '64 mu g/mL'; when the MIC value is the lowest concentration or below, the MIC value is calculated to be less than or equal to 0.125 mu g/mL without distinction. The experiments are all operated in parallel for 3 times, and the MIC value is accepted when the MIC value can be accurately repeated or only differs by one concentration, and the higher concentration is taken as the MIC value; when the MIC values differ by more than two concentrations, re-experiments are required until the requirements are met. The results are shown in Table 1.

TABLE 1 MIC values (μ G/mL) for G-749 for various fungi

As can be seen from Table 1, after the G-749 acts on various fungi for 24 hours, MIC values of 7 fungi (drug-resistant Candida albicans, sensitive Candida albicans, Candida krusei, Candida glabrata, Cryptococcus neoformans, Candida parapsilosis, and Candida tropicalis) are all 16 mug/mL, which shows that the G-749 has a wider antifungal effect.

Example 2 influence of G-749 on the survival of various fungi

The experimental method comprises the following steps:

1. the drugs, strains and materials were as in example 1

2. The experimental steps are as follows:

before experiment, a small amount of bacterial clones are picked from an SDA culture medium stored at 4 ℃, inoculated into a 1mLYEPD culture solution, and subjected to shaking culture at 35 ℃ and 200rpm for 16h, so that the fungi are in the later exponential phase of growth. Diluting with RPMI1640 culture solution, and adjusting the concentration of the culture solution to 1 × 10⁵CFU/mL. Respectively adding fluconazole into the bacterial liquid to ensure that the final concentration is 64 mu G/mL, the final concentration of the compound G-749 is 8 mu G/mL, 16 mu G/mL and 32 mu G/mL, adding DMSO with the same volume into a blank control, and adding D into all bacterial liquidsThe MSO content is less than 0.5%. Performing shaking culture at 200rpm in an incubator at 35 ℃ for 0h and 24h, respectively, sampling each sample, diluting to different concentrations, coating the samples on an SDA plate, performing cloning after culturing for 48 hours, and calculating the survival number of colonies of each group. The results are shown in Table 2.

TABLE 2 number of surviving colonies (CFU/ml) after 7 different fungi were applied to G-749

As shown in Table 2, the colony count of G-749 at a concentration of 32. mu.g/mL after being acted on Candida glabrata, Candida parapsilosis and Cryptococcus neoformans is about 10 from the initial concentration after 24 hours⁵CFU/ml was changed to 10³CFU/mL, which shows that G-749 with the concentration of 32 mug/mL has better growth inhibition effect on the 3 fungi, shows bactericidal activity and has different degrees of bacteriostatic activity on the 7 fungi.

The above experimental results show that G-749 has broad antifungal spectrum and antifungal activity, and thus can be used as antifungal medicine.

Claims (2)

1. Use of G-749 in the preparation of an antifungal medicament, said fungus being Candida krusei (ATCC2340), Candida glabrata (ATCC2281), Cryptococcus neoformans (32609), drug-resistant Candida albicans (103), Candida sensitis (SC5314), Candida parapsilosis or Candida tropicalis.
2. 2. The use according to claim 1, wherein the MIC value of G-749 is 16 μ G/mL.